A GTP affinity probe for proteomics highlights flexibility in purine nucleotide selectivity.

GTP affinity probes are important tools for the study of GTP-binding proteins, and proteomic profiling is a powerful methodology well suited for the study of such a diverse class of proteins. Here, we synthesize and characterize a photoreactive GTP affinity probe that covalently photocross-links to protein targets and has an alkyne handle for click chemistry conjugation to reporter tags. The GTP-BP-yne probe facilitated identification of a variety of GTP-binding proteins by mass spectrometry, such as small GTPases and members of the GTP1/OBG family. Several ATP-binding proteins were also identified, highlighting variability in purine nucleotide selectivity of some proteins, and the probe was used to elucidate targets' relative nucleotide selectivities. The GTP-BP-yne probe will be a useful tool for the study of GTP-binding proteins, especially when targets of interest are not known a priori.